The Use of Genus-Specific Amplicon Pyrosequencing to Assess Phytophthora Species Diversity Using eDNA from Soil and Water in Northern Spain

Abstract

Phytophthora is one of the most important and aggressive plant pathogenic genera in agriculture and forestry. Early detection and identification of its pathways of infection and spread are of high importance to minimize the threat they pose to natural ecosystems. eDNA was extracted from soil and water from forests and plantations in the north of Spain. Phytophthora-specific primers were adapted for use in high-throughput Sequencing (HTS). Primers were tested in a control reaction containing eight Phytophthora species and applied to water and soil eDNA samples from northern Spain. Different score coverage threshold values were tested for optimal Phytophthora species separation in a custom-curated database and in the control reaction. Clustering at 99% was the optimal criteria to separate most of the Phytophthora species. Multiple Molecular Operational Taxonomic Units (MOTUs) corresponding to 36 distinct Phytophthora species were amplified in the environmental samples. Pyrosequencing of amplicons from soil samples revealed low Phytophthora diversity (13 species) in comparison with the 35 species detected in water samples. Thirteen of the MOTUs detected in rivers and streams showed no close match to sequences in international sequence databases, revealing that eDNA pyrosequencing is a useful strategy to assess Phytophthora species diversity in natural ecosystems.

A unique species in Phytophthora clade 10, Phytophthora intercalaris sp. nov., recovered from stream and irrigation water in the eastern USA

“…The self-sterile species P. gallica (Jung & Nechwatal, 2008) and P. intercalaris produce non-papillate and non-caducous sporangia. Although their exact ecological roles remain unknown, these two species have been discovered exclusively from soil and water (Català et al, 2015;Jung & Nechwatal, 2008), which indicates their unique soilborne and waterborne lifestyle.…”

“…The closest species to P. intercalaris, P. gallica, is also a slowly growing species compared with P. gonapodyides, P. lacustris (5Phytophthora taxon Salixsoil) and P. chlamydospora Jung & Nechwatal, 2008), three species frequently recovered from aquatic environments. Isolates of P. gallica were only occasionally recovered, and its presence in streams of northern Spain was only recently confirmed using pyrosequencing (Català et al, 2015). Therefore, whether P. intercalaris is consistently present in irrigation water warrants further investigation, perhaps using different detection methods.…”

“…The pathogenicity of P. gallica has not been fully determined. This species was originally isolated from rhizosphere soil of declining oak and reed stands in France and Germany (Jung & Nechwatal, 2008) and later detected from streams in Spain (Català et al, 2015) and Oregon, USA (Sims et al, 2015). It has been shown to be weakly to moderately aggressive to pedunculate oak (Quercus robur), white willow (Salix alba), common alder (Alnus glutinosa) and European beech (Fagus sylvatica) in pathogenicity tests (Jung & Nechwatal, 2008).…”

Phytophthora Species in Rivers and Streams of the Southwestern United States

“…However, this assumption is now being cast in doubt due to the widespread identification of Phytophthora spp. in streams and forests from across the globe (6)(7)(8)(9)(10)(11)(12)(13). Currently, these species are hypothesized to be waterborne opportunistic pathogens, foliar pathogens, and fine-root or canker pathogens (14).…”

“…A promising new approach for identifying Phytophthora species involves genus-specific amplification and next-generation sequencing of environmental DNA (eDNA). This technique was used to identify 35 species of Phytophthora from Northern Spain, including 13 putative novel species (13).…”